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DNA Replication

DNA Replication. ORC anneals to origin ORC recruits MCM MCM recruits Cdc45p Cdc45p recruits pol a /primase complex RFC displaces pol a and recruits PCNA PCNA recruits pol d DNA ligase stitches DNA fragments together. Regulation of replication. Once and only once Licensing DNA damage

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DNA Replication

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  1. DNA Replication • ORC anneals to origin • ORC recruits MCM • MCM recruits Cdc45p • Cdc45p recruits pola/primase complex • RFC displaces pola and recruits PCNA • PCNA recruits pold • DNA ligase stitches DNA fragments together

  2. Regulation of replication • Once and only once • Licensing • DNA damage • Coordinated with cell cycle • Cyclin • Cyclin dependent kinase (CDK) • Cyclin kinase inhibitor (CKI)

  3. Key regulatory proteins • cdc6/cdt1: licensing agents • E2F/DP1: S-phase transcription factor • Retinoblastoma: E2F repressor • p27/p21 KIP: cyclin kinase inhibitors • p53: cell cycle withdrawal transcription factor

  4. Cell Cycle • G0 • Terminal differentiation • G1 • S • Replication • G2 • M • Division

  5. Cell cycle control • Cyclins • Cell cycle regulated proteins • Cyclin dependent kinases (CDK) • Signaling effectors • Cyclin kinase inhibitors (CKI)

  6. Checkpoint regulation • Phase progression tied to successful completion of prior phase • ALL DNA healthy • ALL DNA replicated • ALL DNA attached to mitotic spindles • Negative/inhibitory regulation • Signal-to-noise • Presence of “No-Go” signal • Threshold of “Ready” signal

  7. Assembly of preRC • ORC, cdc6/cdt1, MCM • Immediately following mitosis • cdt1 • Recruits MCM • Inhibited by geminin • cdc6 • Inhibits MCM helicase • Phosphorylated by CyclinA/CDK2 in S • Translocates to cytoplasm

  8. Initiation of replication • Cyclin A/cdk2 • Releases ORC inhibition • Prevents ORC Re-reformation cdt1 CyA CDK2 cdt1 cdc45 ORC MCM ORC MCM cdc6 cdc6

  9. Licensing • ORC+cdc6 is required to recruit MCM • ORC-cdc6 is required to activate MCM • Cdc6/Cdt1 “licenses” an ORC for replication

  10. Licensing agents • Geminin • cdt1 binding protein • Cell cycle dependent expression • cdt1 • Inhibited by geminin • Stabilized by geminin • Phosphorylated by CyclinA/CDK2 in S • Phospho-form is exported & degraded • Removal allows binding of cdc45

  11. DNA Damage • Base mismatches • Single strand breaks • Double strand breaks • Oxidation/nitrosylation

  12. Strand Break • Non-homologous end joining • Ku mediated recognition of ssDNA • End-to-end repair • Homologous recombination • Rad51 mediated search for homologous template • Template derived patch Ku NHEJ Rad51 HR

  13. Strand Break • ATM kinase recruited to strand break • Ataxia-Telangiectasia Mutated kinase • Autophosphorylates • Phosphorylates H2A • Phosphorylates p53 • p53 • Stabilized and activated by phosphorylation • Activates p21waf/cip (cdk inhibitor) • Blocks transcription of Pold • Blocks transcription of CyclinA

  14. Determination to divide • Integrative • Environmental cues • Systemic/hormonal controls • Internal program

  15. G1 progression • E2F/DP1 transcription factor • DNA polymerase • Cyclins A & E • CDK1 • Retinoblastoma (Rb) • De-phosphorylation dependent E2F binding • Represses E2F/DP1 • Protoconogene • P53 transcription factor • p21 CKI, MDM2

  16. G1 progression • Growth factors cause Rb phosphorylation, which gets degraded, allowing xscription of S-phase proteins • Cdk4/CyD phosphorylates Rb… • Cdk2/CyE phosphorylate RB…. • Cdk2/CyE inhibit p27kip, which inhibits cdk2 • ATM activates p53, which leads to transcription of CKIs p21 & p27

  17. p53/Rb • Active inhibition of cell cycle Phosphorylates to block binding Promotes transcription Cyclin A/E Pol a CDK1 Cyclin D CDK 4/6 Rb E2F S-Phase Binds to block transcriptional activity Mitogens Promotes transcription Inhibits activity Cycle Progression Phosphorylates to stabilize & activate p53 p21CIP/WAF CDK2/4/6 DNA Damage ATM kinase cdc14 phosphatase Dephosphorylates to destabilize & inactivate

  18. Regulatory features • CDKs regulate cell cycle • Phase specific transcription • Cyclin E/cdk2 promotes Cdc6 transcription • Cyclin A/CDK2 activates synthesis • Cyclin B/cdc2 deactivate Mcm • Rb keeps the gate at G1 restriction • Represses CDK2 & polymerase expression • p53 blocks cell cycle & promotes apoptosis • Promotes expression of CKIs

  19. Controls on DNA replication • Growth factors/mitogens • Rb phosphorylation • Cyclin D upregulation • Nutrient availability • Cell size – amino acids, PO4 • GSK inactivates cyclinD • Stress states • DNA damage • ATM phosphorylates p53CKI expression

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